NMR STRUCTURE DETERMINATIONS OF RNA

RNA 的 NMR 结构测定

• 批准号: 6169731
• 负责人: ARTHUR PARDI
• 金额: $ 20.91万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6169731
• 关键词: animal genetic material tag; genetic regulatory element; high performance liquid chromatography; human genetic material tag; messenger RNA; nuclear magnetic resonance spectroscopy; nucleic acid structure; oligonucleotides; ribozymes

This proposal will continue our program on NMR structural studies of RNA. Although there has been a huge increase in our understanding of the various functions that RNAs perform in cells, there is still relatively little information on the three-dimensional structures of RNA. Part of this project involves development of improved NMR methods for resonance assignment and solution structure determinations of RNA oligonucleotides. The ability to generate uniformly 13C/15N labeled RNAs of defined sequence has allowed powerful 2D, 3D and 4D heteronuclear NMR experiments to be applied to nucleic acids. Some areas that will be studies include: application of network-editing NOESY experiments for eliminating certain types of spin-diffusion artifacts; improved methods for extracting coupling constants in isotopically labeled RNAs; probing the dimerization/aggregations state of RNAs by NMR measurements of translational diffusion; and methods for improving the yields and purification of RNAs by use of ribozymes as site-specific RNA cleavage reagents. 13C relaxation experiments will be used to probe the dynamics of several Rna structural motifs, including a CUUG RNA tetraloop. The power dependence of the T1p will be measured for 1H, 13C, 15N and 31P resonances in this tetraloop in order to probe slower motions on the mus to ms timescale. NMR will be used to probe the structure and synamics of the iron response element (IRE) RNA. This RNA motif is located at the 5' untranslated region of all ferritin mRNAs and the 3' untranslated region of transferrin receptor mRNA. In conjunction with the iron response element binding protein, the IRE RNA helps regulate iron levels in a cell by controlling the translation of iron import and storage proteins. Multi-dimensional heteronuclear NMR techniques will be used to determine the three-dimensional structure of the IRE RNA several mutant IRE RNAs that have been identified through in vitro selection experiments.

这项建议将继续我们的核磁共振结构研究计划 核糖核酸。尽管我们的理解有了很大的提高 在RNA在细胞中执行的各种功能中，仍然有 关于RNA三维结构的信息相对较少。 该项目的一部分涉及改进的核磁共振方法的开发。 核糖核酸的共振指认和溶液结构测定 寡核苷酸。能够生成统一的13C/15N标签 已定义序列的RNA允许强大的2D、3D和4D 异核核磁共振实验将应用于核酸。一些人 将研究的领域包括：网络编辑的应用 消除某些类型自旋扩散的NOESY实验 人工产物；用于提取耦合常量的改进方法 同位素标记的RNA；探测二聚化/聚集态 通过平移扩散的核磁共振测量获得RNA；以及 提高核糖核酸产量和纯度的方法 核酶作为位点特异性RNA裂解试剂。13C松弛 实验将被用来探索几种RNA的动力学 结构基序，包括CUUG RNA四环。那股力量 将测量1H、13C、15N和31P的T1p的依赖关系 在这四圈的共振，以便探测较慢的运动 Mus to ms TimeScale。将使用核磁共振来探测结构和 铁反应元件(IRE)RNA的动力学。这个RNA基序 位于所有铁蛋白mRNAs的5‘非翻译区， 转铁蛋白受体基因3‘端非翻译区。结合 通过铁反应元件结合蛋白，IRE RNA有助于 通过控制铁的转化来调节细胞中的铁水平 进口和储存蛋白质。多维异核核磁共振 这些技术将被用来确定 已鉴定的几个突变的IRE RNA 通过体外筛选实验。